This disclosure relates generally to screening compounds for binding with targets, including drug targets, and more specifically to the differentiation of binding at different sites on targets, including drug targets, an example being differentiation of inhibitory binding at a specific substrate site and inhibitory binding at some other substrate, cofactor, regulator, or signaling site on an enzyme target.
Molecules that can inhibit or otherwise modify the activity of a drug target can be good drug candidates or leads, so there is a great deal of interest in screening compound libraries against drug targets to find such leads. For example, molecules that bind to an enzyme at a site that would otherwise bind a substrate or cofactor can slow down the reaction or even prevent the substrate from reacting. Additionally, a lead molecule that binds to an allosteric regulatory site can modify the enzymatic process, which could also be useful for therapeutic purposes. However, a good drug candidate also needs to be specific to the enzyme being targeted; it is generally not good therapeutically for a drug candidate to affect many different enzymes in a large class.
Enzymes, which are biomolecules that catalyze reactions between other molecules and are key to biological function, constitute one class of drug targets. In the enzymatic process, the enzyme binds the reacting molecule or molecules at specific binding sites, the case of two reacting molecules being the most common. For example, ATP kinases catalyze reactions between ATP (adenosine 5′-triphosphate) and a second, enzyme-specific substrate, resulting in a phosphorylated product and ADP (adenosine 5′-diphosphate). An enzyme may also bind cofactors that act to enable the enzyme function, as well as binding other inhibitors, effectors, or signaling partners. Thus, it is often the case that enzymes have more than one binding site that affects the rate of catalysis, including substrate sites, one or more cofactor sites, and other regulatory or signaling sites.
One way to screen for leads that are specific to a particular enzyme is to target a specific site on the enzyme that binds a substrate, cofactor, regulator, or signaling partner that is largely specific to the enzyme. As an example of this strategy, consider screening for binding to a protein kinase. ATP-dependent protein kinases catalyze reactions between ATP and a protein substrate, with the particular protein substrate being different for different protein kinases. Since ATP binding is common to all ATP kinases, a test compound that binds solely to the ATP-binding site on a target enzyme has a significant chance of binding to many other kinases. On the other hand, a test compound that binds to the same site as the protein substrate for that enzyme is a candidate for being more specific to that enzyme target. In this example, one would screen for binding to the same site as the protein substrate. This example can be extended to other kinases (e.g., non-protein substrates, ADP-dependent kinases and other non-ATP kinases) as well. Examples of targets other than kinases with multiple binding sites include G-proteins, which all hydrolyze GTP (guanosine 5′-triphosphate) but transfer signals between enzyme-specific GPCR (G-protein coupled receptor) regulators and downstream signaling targets; monomeric GTPases, which all hydrolyze GTP but signal specific molecules; and dehydrogenases, including those that use NAD+ (nicotinamide adenine dinucleotide), NADP+ (nicotinamide adenine dinucleotide phosphate), or FAD (flavin adenine dinucleotide) to dehydrogenate a second, enzyme-specific substrate.
More broadly, many proteins in any given family often share a common substrate, cofactor, regulator, or other ligand (such as an upstream or downstream signaling partner), while also having a different substrate, cofactor, regulator, or other ligand that is more specific to the protein of interest. Targeting the binding sites of the more specific ligands on protein drug targets can be useful in drug discovery.
In drug discovery today with enzyme targets, an endpoint assay is often used to determine if an enzymatic reaction has gone to completion, and in the initial high-throughput screening phase, as many as millions of drug candidates are screened using this type of assay. In such a screen, an inhibitor is added to the enzyme-cofactor-substrate mixture, and the mixture is tested for reaction. Today, the majority of high-throughput assays are designed to detect the converted substrate, i.e., they detect the endpoint of the enzymatic process (such as in the widely-used antibody-binding assays for protein phosphorylation). Such assays do not easily differentiate between binding of inhibitors at different sites. While follow-up kinetic studies and titration measurements of inhibitor activity can help differentiate between inhibitors that compete with a particular ligand for binding and those that do not, there is a pressing need to develop high-throughput methods that can easily and directly discriminate between binding at different binding sites, including as an example discriminating between competitive inhibition at different binding sites.
The need for high-throughput methods has increased in recent years as researchers and companies have turned to combinatorial methods and techniques for synthesizing, discovering and developing new compounds, materials, and chemistries. For example, pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. See Handbook of Drug Screening (R. Seethala and P. B. Fernandes, eds., Marcel Dekker Inc., 2001) for a review of progress in this area. The recent explosion in the number of potential drug targets due to the accelerated implementation of genomics technologies and the completion of the Human Genome sequence has only increased the need to develop effective high-throughput methods for screening against drug targets. Combinatorial methods are also being applied to other industries, as illustrated by companies such as Symyx Technologies® that is applying combinatorial techniques to materials discovery in the life sciences, chemical, and electronics industries.
To further illustrate the use of combinatorial chemistry methods and the need for improved methods, we now discuss the example of pharmaceutical research in this area in more detail. Pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. A combinatorial library is a collection of chemical compounds that have been generated, by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” as reagents. For example, a combinatorial polypeptide library is formed by combining a set of amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can theoretically be synthesized through such combinatorial mixing of chemical building blocks.
Once a library has been constructed, it must be screened to identify compounds which can be used as leads to design drug candidates possessing some kind of biological or pharmacological activity. For example, screening can be done with a specific biological compound, often referred to as a target, which participates in a known biological pathway or is involved in some regulatory function. The library compounds that are found to react with the targets are candidates for affecting the biological activity of the target, and hence can be useful leads for developing a candidate for a therapeutic agent.
Since combinatorial methods involve looking at a large number of compounds and reactions in parallel, there is a need for tools that can measure reactions and interactions of large numbers of small samples in parallel, consistent with the needs of combinatorial discovery techniques. Preferably, users desire that these tools enable inexpensive measurements and minimize contamination and cross-contamination problems.
One method for measuring reactions and interactions is calorimetry. Calorimetry can be used to measure the thermodynamics and kinetics of reactions without requiring that reactants be labeled (e.g., radio-labeled or labeled with fluorophores) or immobilized on surfaces. Most other current methods require some modification of either the substrate or a cofactor (fluorescent labeling, surface anchoring, etc.) [Handbook of Drug Screening, R. Seethala and P. B. Fernandes, eds., Marcel Dekker Inc., 2001]. These modifications add steps and cost to an assay, and they can potentially modify the reagents in undesired ways that may not be understood at the time of an assay. Furthermore, it would be useful to have a method capable of replacing the cost of antibodies that are used in many enzyme endpoint assays, as it is largely the single largest cost contributor to overall screening cost. Calorimetry does not rely on antibody binding to products of enzymatic reactions.
In some cases, the sample to be studied is precious, and it might not be acceptable to use the relatively large amount of material required by a standard microcalorimeter to perform only one measurement. For example, one may desire to study a natural extract or synthesized compound for biological interactions, but in some cases the available amount of material at concentrations large enough for calorimetry might be no more than a few milliliters. Performing a measurement in standard microcalorimeters, such as those sold, for example, by MicroCal® Inc. (model VP-ITC) or Calorimetry Sciences Corporation® (model CSC-4500), requires about 1-2 ml of sample, which means that one would possibly be faced with using a majority or all of the precious material for one or a small series of measurements. Tools that enable calorimetric measurements with much smaller sample sizes would be helpful in overcoming this limitation. Furthermore, standard microcalorimeters require hours for one measurement, whereas high-throughput screening requires orders of magnitude higher throughput.
In drug development activities, it is sometimes the case that a strongly binding drug lead was discovered for a particular drug target, only to have it later determined that the drug lead is not specific enough to the target or has other drawbacks that render it not useful for further drug development. In such cases, investigators may chose to reinvestigate the drug target and look for drug hits or leads that bind to sites on the target other than the binding site for the previously discovered drug lead. As an example, investigators have sometimes found inhibitors of ATP kinases that are strong binders to the ATP binding site, only to learn that the inhibitors are not sufficiently specific to the kinase of interest, or otherwise bind or interact with other molecules in a way that produces adverse effects. One approach that an investigator may take in such a case is to reinvestigate the drug target to find drug hits or leads that bind to sites other than the ATP binding site. There is a need for high-throughput screening methods that allow investigators to screen for binding to a site other than the binding site of a previously discovered strongly binding ligand. In particular, there is a need for such methods that are also generic in the sense that they do not require labeling (e.g., fluorescent, chemiluminescent, or radio-labeling) of compounds, immobilization of compounds on surfaces, or other assay-specific modifications of the molecules being studied.